The invention concerns new complex compounds for the diagnosis of vascular diseases, processes for their production as well as diagnostic agents containing these compounds.
Arteriosclerosis is a chronic, progressive disease of the blood vessels, which previously could only be clinically diagnosed at an advanced stage. Vessel change caused through arteriosclerosis are today conventionally determined through arteriography. Here a contrasting agent is applied via a catheter into the vessel of interest and stenosis of the regions of the vessel is detected through X-rays. A disadvantage of this procedure is that only partial regions of the vascular system can be observed. Because arteriography is an invasive procedure, complications can arise upon its application like for example pain, perforation of an artery, arrhythmia, heart attack or stroke, and under unfortunate circumstances even death of the patient.
Furthermore procedures based on the application of ultrasound as well as MR-tomography are also used for the diagnosis of arteriosclerosis.
All the presently used procedures have the great disadvantage in that the arteriosclerotic vessel change is detected through the observation of contracted blood flow or significant changes in the artery wall and therefore detected only at an advanced stage of atherogenesis.
Early detection of arteriosclerosis would be of great significance for example for the control of therapeutical effect of diet, calcium antagonists, lipid- and high blood pressure reducers, the control of restenosis after angioplasty and the diagnosis of coronary heart diseases as well as the detection of thrombotic vessel deposition.
Non-invasive techniques for the diagnosis of arteriosclerosis have been described. Radioactive labeled antibodies or also labeled low density lipoproteins (LDL) are introduced which bind on the arteriosclerotic wall regions (Lees et al. 1993, J. Nucl. Med. 24, 154-156; Kaliman et al. 1985, Circulation, 72, 300; Virgolini et al. 1991, Eur. J. Nucl. Med., 18, 944-947). These methods however have decisive disadvantages, for example the antigenic effect of the antibodies on the organism and the long duration (several days) necessary for the isolation, purification and labelling of LDL from the patient""s blood. These large molecules also reveal a long half life in the blood which together with a high background radiation over the whole body making the localization of arteriosclerotic lesions difficult if not impossible.
Shih et al. (1990, Proc. Natl. Acad. Sci., 87, 1436-1440) have synthesized partial sequences of the LDL-protein portions (apo-B-100), which although still bind to arteriosclerotic plaque, reveal an overall shorter half-life in blood and a better signal-noise ratio. Due to their low affinity to plague and/or the low density of binding sites for this apo-B-peptide in the plaque, a successful in vivo diagnosis of arteriosclerosis could not be established.
The object of the invention is to provide new compounds and agents which are suitable for non-invasive diagnostic procedures, especially for the diagnosis of early, but not yet stenotic, vascular diseases.
Endothelines are physiologically active peptides, which perform hormonal as well as neuroregulatory functions in organisms (MacCumber et al. 1989, Proc. Natl. Acad. Sci., 7285-7289; Yanagisawa et al. 1989, Trends Pharmacol. Sci., 10, 374-378; LeMonier die Gouville et al., 1989, Life Scd., 45, 1499-1513; Yanagisawa et al., 1988, Nature, 332, 411-415). Up to now four different isotypes have been identified in humans (Inoue et al., 1989, Proc. Natl. Acad. Sci., 86, 2863-2867). Endotheline 1 is a polypeptide having the following sequence of 21 amino acids:
Cys-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val-Tyr-Phe-Cys-His-Leu-Asp-Ile-Ile-Trp (Yanagisawa et al., 1988, Nature, 332, 411-415).
From vascular endothelium an inactive precursor of the endotheline, the Big Endotheline, is formed. After a heptadecapeptide is splitted off through the Endotheline-Converting-Enzyme (ECE) endotheline originates, which binds on specific receptors of smooth vascular musculature. There it leads to a Ca++-facilitated contraction of the smooth muscle cells (Yanagisawa et al., Nature, 332, 411-415; Takuwa et al. 1991, Contrib. Nephrol., 90, 99-104).
One of the early irreversible changes arising from atherogenesis is, among others, the proliferation of smooth muscle cells of the vessel wall brought about by the growth factor (e-g. PDGF) (Desmoulixc3xa8re and Gabbiani 1992, Cerebrovasc. Dis., 2, 63-71). Through in vitro incubation of human atheromatotic coronary arteries with 125I-endotheline 1, it could be shown that an increased binding of 125I-endotheline 1 in the region of Tunica media as well as Vasa vasora region appears (Dashwood et al., 1991, J. Cardiovasc. Pharmacol., 17, 458-462). Through the application of 125I-endotheline 1 in rabbits, whose abdominal aorta were previously de-endothelisized via a balloon catheter, an increased uptake of 125I-endotheline 1 in the de-endothelisized regions of the aorta could be shown, which indicates a greater density of binding sites for endotheline 1 in the damaged vessel regions (Kurata et al. 1992, J. Nucl. Med., 33, 845). From this investigation it can be concluded that the proliferated smooth muscle cells also further express endotheline receptors.
Endotheline 1 has a strong vasoconstrictory effect on smooth vascular musculature (A. M. Doherty, 1992, Medical Chemistry, 35, 1493-1508). Therefore, it can only be applied i.v. relative low concentrations into the organism. Higher concentrations can only be applied with partial sequences of endothelines, endotheline derivatives, endotheline analogs or endotheline antagonists, which although bind to the endotheline receptor, but do not lead to such a marked contraction of the smooth muscle cells.
Since endothelines are eliminated very quickly through the kidneys, disturbing background radiation caused by the uptake of endothelines by other organs or tissues is extremely low.
The German patent application P 43 01 871.8 already describes compounds containing endothelines, partial sequences of endothelines, endotheline derivatives, endotheline antagonists or endotheline analogs, which bind to endotheline receptors.
During the search for additional compounds the substance was found which has the general formula I
(K)z-(L)y-(A1)aa-(A2)bb-(A3)cc-(A4)dd-(A5)ee-(A6)ff-(A7)gg-(A8)hh-(A9)jj-(A10)kk-(A11)ll-Ile-Ile-Trpxe2x80x83xe2x80x83(I)
in which
aa, bb, cc, dd, ee, ff, gg, hh, jj, kk and ll stand for, independent from one another, the integers 0, 1 or 2,
y and z represent independent from one another the integers 0 or 1,
K depicts a chelate-forming residual having the general formula IIA or IIB 
in which
T is a hydrogen atom or a bond at a metal atom or a suitable sulfur protecting group like an alkali metal ion, a C1-6-acyl residual, a benzoyl residual, a hydroxyacetyl residual, an acetamidomethyl residual, a p-methoxybenzyl residual, an ethoxyethyl residual, a benzyl residual, an ortho- or para-hydroxybenzyl residual, an ortho- or para-acetoxybenzyl residual, a p-nitrobenzyl residual, a 4-picolyl residual, a 2-picolyl-N-oxid residual, a 9-anthrylmethyl residual, a 9-fluorenyl residual, a ferrocenylmethyl residual, a diphenylmethyl residual, a bis(4-methoxyphenyl)methyl residual, a dibenzosuberyl residual, a triphenylmethyl residual, a diphenyl-4-pyridylmethyl residual, a phenyl residual, a 2,4-dinitrophenyl residual, a tert-butyl residual, a 1-adamantyl residual, a methoxymethyl residual, a isobutoxymethyl residual, a 2-tetrahydropyranyl residual, a benzylthiomethyl residual, a phenylthiomethyl residual, a trimethylacetamidomethyl residual, a benzamidomethyl residual, an acetylmethyl residual, a carboxymethyl residual, a cyanomethyl residual, a 2-nitro-1-phenylethyl residual, a 2-(4xe2x80x2-pyrdyl)ethyl residual, 2-cyanoethyl residual, a 2,2-bis(carboethoxy)ethyl residual, a 1-m-nitrophenyl-2-benzoylethyl residual, a 2-phenylsulfonylethyl residual, a 1-(4-methylphenylsulfonyl)-2-methylprop-2-yl residual, a silyl residual, a N-{[(p-biphenyl)isopropoxy]carbonyl}-N-methyl-xcex3-aminobutyrate residual, a N-(tert.-butoxycarbonyl)-N-methyl-xcex3-aminobutyrate residual, a 2,2,2-trichlorethoxycarbonyl residual, a tert.-butoxycarbonyl residual, a benzyloxycarbonyl residual, a p-methoxybenzyloxycarbonyl residual, a N-ethyl residual, a N-methoxymethyl residual, a S-ethyl residual, a tert.-S-butyl residual, a substituted S-phenyl residual, a sulfonate residual, a sulfenylthiocarbonate residual or a 3-nitro-2-pyridinsulfenyl residual, or whereby two T residuals form together with those attached S atoms inter- or intramolecular disulfide bridges,
R4 stands for a residual having the formula IIC or IID 
where
the *-marked carbon atoms are bound to the imino group of the formula IIB and
nxe2x80x2 stands for the integers 1 or 2,
i for any number from 2 to 6, and TT for residuals of xcex1-amino acids like alanine, valine, leucine, isoleucine, proline, tryptophane, phenylalanine, methionine, glycine, serine, tyrosine, threonine, cysteine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, citrulline, homocysteine, homoserine, 4-hydroxyproline, 5-hydroxylysine, ornithine, sarcosine or taurine and/or their homologous xcex2-amino acids, which are bonded in the usual way over the amide groups,
as well as chelate forming residuals having the formula IIE 
in which
R6 represents a hydrogen or a C1-6-alkyl residual,
as well as chelate forming residuals having the formula IIF 
in which
the residuals R7 to R18 each represent independent from one another a hydrogen atom, a C1-10-alkyl chain and/or a bond on L, and o, p and r mean each the numbers 1 or 2 and T has the above given meaning,
as well as chelate forming residuals having the formula IIG 
in which
R19 to R24 are equal or different and depict independent from one another a hydrogen atom or a C1-4-alkyl residual,
mxe2x80x2 stands for the integers 2 or 3,
as well as chelate forming residuals having the formula IIH 
in which
X1 means a bond, a methylene group or a CHY4-group, and one of the groups Y1, Y2, Y3 or Y4 stands for a bond to L and the remaining indicate hydrogen or optionally depict an oxygen atom,
T has the above given meaning, and
A1, A2, A3 and A4 are equal or different and represent independent from one another hydrogen or a C1-6-alkyl residual,
as well as chelate forming residuals having the formula IIJ 
in which
R27 depicts a hydrogen atom or optionally a C1-6-alkyl residual substituted with one or two hydroxyl groups,
R25 and R26 represent each a hydrogen atom or together an oxygen atom,
A a hydroxyl- or mercapto group,
Y a hydrogen atom, a carboxy- or sulfonyl residual and
Z a carbon atom or a nitrogen atom,
as well as chelate forming residuals having the formula IIK,
(AA1)mm-(AA2)nn-(AA3)oo-(AA4)pp-(AA5)qq-(AA6)rr(IIK)
in which
mm, nn, oo, pp, qq and rr stand for, independent from one another, the integers 0, 1 or 2,
AA1 to AA6 stand for, independent from one another, residuals of xcex1-amino acids like alanine, valine, leucine, isoleucine, proline, tryptophane, phenylalanine, methionine, glycine, serine, tyrosine, threonine or cysteine, which optionally bears a T group with the above given meaning instead of the hydrogen atom on the sulfur, asparagine, glutamine, asparatic acid, glutamic acid, lysine, arginine, histidine, citrulline, homocysteine, homoserine, 4-hydroxyproline, 5-hydroxylysine, ornithine, sarcosine or taurine and/or their homologous xcex2-amino acids, which are bonded in the usual way over the amide groups, and whereby optionally each two available cysteine residuals are linked to one another to form one cystine residual,
as well as chelating substances having the formula II L, 
in which
Ra, Rb, Rc, Rd, Rf, Rg and Rh are equal or different and each stands for a hydrogen atom and/or a branched or non-branched C1-C6-alkyl residual,
Re depicts a carboxy group or a non-branched, branched, cyclic or polycyclic Cl-C60-alkyl-, alkenyl-, polyalkenyl-, alkinyl-, polyalkinyl-, aryl-, alkylaryl- or arylalkyl residual, which is optionally substituted with hydroxy-, oxy-, oxo-, carboxy-, aminocarbonyl-, alkoxycarbonyl-, amino-, aldehyd- or alkoxy groups having up to 20 carbons atoms and/or optionally through one or more heteroatoms from the series O, N, S, P, As, Se intermitted and/or substituted,
T has the above given meaning,
L stands for a bond or a hydrogen atom or a non-branched, branched, cyclic or polycyclic C1-C60-alkyl-, alkenyl-, polyalkenyl-, alkinyl-, polyalkenyl, aryl-, alkylaryl- or arylalkyl residual, which is optionally substituted with one or more hydroxy-, mercapto-, alkylmercapto-, oxo-, oxy-, carboxy-, aminocarbonyl-, alkoxycarbonyl-, amino-, aldehyd- or alkoxy groups having up to 20 carbon atoms and/or optionally through one or more heteroatoms from the series O, N, S, P, As, Se intermitted and/or substituted,
or depicts a Z1xe2x80x94Rxe2x80x94Z2 residual,
in which
R stands for a straight chain, branched, saturated or unsaturated C1-20 alkyl residual which is optionally intermitted through one or more oxygen and/or sulfur atoms and/or carbonyl-, xe2x80x94NHCOxe2x80x94, xe2x80x94N(C1-6-alkyl)COxe2x80x94, xe2x80x94NHxe2x80x94 and xe2x80x94N(C1-6-alkyl) residuals and optionally substituted with hydroxy- and/or epoxy groups,
Z1 and Z2 independent from one another, each is an xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94(Cxe2x95x90O)Oxe2x80x94, xe2x80x94NH(Cxe2x95x90S)NHxe2x80x94, xe2x80x94(Cxe2x95x90O)xe2x80x94 or xe2x80x94NH(Cxe2x95x90S)xe2x80x94 group,
or stands for a residual having the formula xcex1
in which
s and t stand for independent from one another the integers 0, 1, 2 or 3, the ring B indicates a phenyl- or cyclohexyl residual and Z1 and Z2 have the above given meaning,
A1 to A11 represent independent from one another L-xcex1- or D-xcex1-amino acid residuals, xe2x80x94NHxe2x80x94CH(Z)xe2x80x94COxe2x80x94 residuals,
in which
Z has the meaning
Hxe2x80x94, CH3xe2x80x94, (CH3)2CHxe2x80x94, (CH3)2CHxe2x80x94CH2xe2x80x94,
CH3xe2x80x94CH2xe2x80x94CH(CH3)xe2x80x94, CH3xe2x80x94Sxe2x80x94(CH2)2xe2x80x94, HOOCxe2x80x94CH2xe2x80x94,
HOOCxe2x80x94(CH2)2xe2x80x94, H2Nxe2x80x94(CH2)3xe2x80x94, H2Nxe2x80x94C(xe2x95x90NH)xe2x80x94(CH2)3xe2x80x94,
HOxe2x80x94CH2xe2x80x94, CH3xe2x80x94CH(OH)xe2x80x94, HSxe2x80x94CH2xe2x80x94, HSxe2x80x94(CH2)2xe2x80x94,
H2Nxe2x80x94COxe2x80x94CH2xe2x80x94, H2Nxe2x80x94COxe2x80x94(CH2)2xe2x80x94, (HOOC)2CHxe2x80x94CH2xe2x80x94, 
in which n means the integers 0, 1 or 2,
or residuals 
or taurine residuals,
as well as their complexes with metal ions having atomic numbers 21-32, 37-39, 42-51 and 57-83 and their water soluble salts,
which surprisingly enrich the arteriosclerotic plaque to an even stronger extent, and because of low background radiation in other tissues and organs resulting from the quick elimination of these substances, they are preferentially suited for non-invasive diagnosis of arteriosclerosis.
Particularly preferred compounds of the general formula I according to the invention are Asp-Gly-Gly-Cys-Gly-Cys-His-Leu-Asp-Ile-Ile-Trp, Asp-Gly-Gly-Cys-Gly-cys-D-Trp-Leu-Asp-Ile-Ile-Trp, Asp-Gly-Gly-Cys-Gly-Cys-Phe-(D-Trp)-Leu-Asp-Ile-Ile-Trp, N-(4-hydroxycarboxy-1-oxo-3-thia-but-1-yl)-Cys-Phe-(D-Trp)-Leu-Asp-Ile-Ile-Trp, N-(4-hydroxycarboxy-1-oxo-3-thia-but-1-yl)-Cys-(D-Trp)-Leu-Asp-Ile-Ile-Trp, N-(4-hydroxycarboxy-1-oxo-3-thia-yl)-Cys-His-Leu-Asp-Ile-Ile-Trp, N-(mercaptoacetyl)-Gly-Gly-Asp-Phe-(D-Trp)-Leu-Asp-Ile-Ile-Trp and N-(mercaptoacetyl)-Gly-Gly-Asp-(D-Trp)-Leu-Asp-Ile-Ile-Trp.
Particularly preferred complex compounds according to the invention are Tc-99m-complex of Asp-Gly-Gly-Cys-Gly-Cys-His-Leu-Asp-Ile-Ile-Trp, Tc-99m-complex of Asp-Gly-Gly-Cys-Gly-Cys-D-Trp-Leu-Asp-Ile-Ile-Trp, Tc-99m-complex of Asp-Gly-Gly-Cys-Gly-Cys-Phe-(D-Trp)-Leu-Asp-Ile-Ile-Trp, Tc-99m-complex of N-(4-hydroxycarboxy-1-oxo-3-thia-but-1-yl)-Cys-Phe-(D-Trp)-Leu-Asp-Ile-Ile-Trp, Tc-99m-complex of N-(4-hydroxycarboxy-1-oxo-3-thia-but-1-yl)-Cys-(D-Trp)-Leu-Asp-Ile-Ile-Trp, Tc-99m-complex of N-(4-hydroxycarboxy-1-oxo-3-thia-but-1-yl)-Cys-His-Leu-Asp-Ile-Ile-Trp, Tc-99m-complex of N-(mercaptoacetyl)-Gly-Gly-Asp-Phe-(D-Trp)-Leu-Asp-Ile-Ile-Trp and Tc-99m-complex of N-(mercaptoacetyl)-Gly-Gly-Asp-(D-Trp)-Leu-Asp-Ile-Ile-Trp.
It was found that the radioactive labelled metal complexes of the general formula I according to the invention concentrated surprisingly strongly at arteriosclerotic vascular lesions and thus reach a sufficient concentration for display with a scintillation camera or other suitable apparatus common in nuclear medicine. Surprising was the finding that the applied substances according to the invention reach this concentration in vivo so fast and the bonding is so stable that even after removal and elimination of the excess substance binding to the receptors, a high and sufficient concentration for diagnosis remains. Furthermore it was surprising that the enrichment takes place preferable at altered, through the most various of ways, artery wall regions which are to be diagnosed, even though endotheline itself is effective on all vascular areas.
The applied substances according to the invention possess the additional advantage that in contrast to many other substance classes and substances tested, they do not concentrate additionally and nonspecifically in other tissues and organs which is decisive for suitablility as a diagnostic.
Scintigrams show the enrichment in vivo of the compounds according to the invention.